Infections with Shiga toxin-producing Shigella dysenteriae type I or Shiga-like toxin-producing E. coli cause outbreaks of dysenteric disease which continue to be a major cause of morbidity and mortality in maid parts of the world. Patients infected with these toxin-producing bacteria are at increased risk for developing life-threatening complications involving extensive damage of blood vessels serving the renal and central nervous systems. We have previously shown that purified Shiga-like toxins (SLTs) only manifest direct cytotoxicity for human vascular endothelial cells when present at high doses. We hypothesized that both SLTs and cytokines produced in response to the toxins or other bacterial products were necessary to cause the profound vascular lesions characteristic of the toxin-associated diseases. We have shown that murine peritoneal macrophages are essentially refractory to the cytotoxic actions of SLTs but respond to toxins by producing proinflammatory cytokines (TNF-alpha, IL-1alpha, IL-6) in vitro. We now wish to extend our examination of SLT- mediated cytokine production to human peripheral blood monocytes, human macrophage-like cell lines, and human renal epithelial cells. Use of macrophage-like cell lines allows us to treat the cells with differentiation factors to examine the relationship of cell maturation with toxin sensitivity, toxin receptor expression, and cytokine production. We will examine the SLT holotoxin structural component(s) and biological activity(ies) necessary to mediate cytokine production. We will use a series of inhibitors of transmembrane signalling pathways to determine the mechanism(s) of SLT-induced cytokine expression. We will utilize a well-defined mouse model of SLT-mediated kidney damage to measure in vivo cytokine mRNA and product expression in response to challenge with SLT-producing coli or injection of purified SLTs. The levels of cytokines will then be modulated in toxin-treated animals to examine the role of cytokines in kidney damage and death. Finally, we will infuse purified SLTs into baboons to define, clinically and histopathologically, a large animal model of SLT-mediated diarrhea and acute renal failure.